Contents - Faperta

470 Biotechnological Approaches for Pest Management and Ecological Sustainability
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3 4 5 6 7 8 9 10 11 12 C +ve M
FIGURE 16.1 RT-PCR analysis of genetically engineered pigeonpea plants for cry1Ac gene. Lanes 1 to 12 are
pigeonpea transgenic events with cry1Ac gene, lane C is a negative control, lane positive is the plasmid DNA
having the cry1Ac gene, and lane M is the 1 kb ladder as marker.
of the DNA to be analyzed is prepared, and a constant amount of the competitor added.
After completion of the PCR, the resulting amplifi cation products are visualized through gel
electrophoresis. When both DNA targets yield the same amount of product, it is assumed
that the starting amount was also the same. By setting up two competitive PCRs, one for
the transgenic crop (Bt maize) and one for the species of interest (nontransgenic maize),
and including competitors in both, the quantity of transgenic crop relative to the species
can be estimated by extrapolation from the degree of dilution and concentration of the
competitors. The competitive PCR methods are semiquantitative.
Real-time PCR is based on continuous monitoring of PCR product. This is done via
fl uorometric measurement of an internal probe during the reaction. In real-time analyses,
the amount of product synthesized during PCR is estimated directly by measuring
fl uorescence in the PCR reaction. Several types of hybridization probes are available that
emit fl uorescent light corresponding to the amount of synthesized DNA. The quantitative
estimation is based on extrapolation by comparing the transgenic crop sequence relative to
the reference, for example, gene sequence cry1Ac (Figure 16.1) and ivr1 gene from maize.
With the use of fl uorescence, it becomes possible to measure exactly the number of cycles
that are needed to produce a certain amount of PCR product. Since one cycle corresponds
to doubling the amount of product, a simple formula can be used to estimate the ratio.
While real-time PCR requires more sophisticated and expensive equipment than competitive
PCR, it is faster, automated, and more specifi c. Presently, real-time PCR can be considered
as the most powerful tool for the detection and quantifi cation of food derived from
transgenic crops.
Detection systems for monitoring the presence of genetically modifi ed food could also
be based on the presence of the 35S promotor, which has been used in many insect protected
crops. However, the detection limit based on 35S has been found to vary by a factor
of 20 in different laboratories, and is not suited to distinguish between genetically modifi
ed food mixers and co-mingling of produce during harvest, transport, and storage.
It allows the detection of 35S promotor in the range of 0.01% to 0.1%. The fi xing of a limit of
1% as the basis for labeling food as genetically modifi ed has necessitated the use of quantitative
PCR (Hardegger, Brodmann, and Herrmann, 1999), and real-time PCR (Heid et al.,
1996). Hubner, Studer, and Luthy (1999) suggested that QC-PCR could be used for survey
of threshold values of 1% for the labeling of genetically modifi ed food.
Microarray Technology
In this technique, many selected probes are bound in an array format to a solid surface,
with each spot containing numerous copies of the probe. The array is then hybridized
with the isolated DNA sample of interest labeled with a fl uorescent marker. During the
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